The Mechanism Of Bazheng Powder In Treating Bacterial Urinary Tract Infection In Children Based On Bacterial Adhesion

Urinary tract infection is a common infectious disease.It is one of the most common infectious diseases in children.Clinically,uropathogenic Escherichia coli(UPEC)is the main pathogen causing urinary tract infection.The main virulence factors of UPEC are fimbriae,hemolysin,iron acquisition system.Fimbriae is the most important virulence factor of UPEC.Fimbriae of UPEC mainly include type 1 fimbriae,P fimbriae and curli fimbriae.Fimbriae mediates various functions of bacteria.Among them,fimbriae adhesion to the surface of urothelium is the first step for UPEC to colonize the urinary tract.Bazheng powder is a representative formula for the treatment of damp heat drenching syndrome.Clinically,it is mainly used to treat urinary tract infection caused by damp heat infusion.Starting from the adhesion function of UPEC fimbriae.Based on bacterial adhesion,this study discussed the antibacterial mechanism of Bazheng powder on UPEC from two aspects of bacteria and host,so as to clarify the mechanism of Bazheng powder in the treatment of urinary tract infection.1 The antibacterial effect of Bazheng Powder on uropathogenic Escherichia coliObjective: Explore the antibacterial effect of Bazheng Powder on UPEC,Construct an in vitro model of urinary tract infection,and explore the antibacterial effect of Bazheng Powder on UPEC from the in vitro model of urinary tract infection.Methods: The macro broth dilution method was used to determine the minimal inhibit concentration of Bazheng Powder on UPEC,and then a growth curve was drawn to determine the influence of Bazheng Powder on the growth curve of UPEC.Use a soft agar plate to detect the effect of Bazheng San on the gliding motility of UPEC.The CCK-8method was used to detect the cytotoxicity of Bazheng Powder to T24 cells.At a concentration lower than cytotoxicity,adhesion test and invasion test were used to determine the effect of Bazheng Powder on the adhesion and invasion ability of UPEC.Using transmission electron microscope to observe the influence of Bazheng Powder on the structure of fimbriae and flagella of UPEC.Crystal violet staining and Confocal laser scanning microscope were used to detect the effect of Bazheng Powder on the formation of UPEC biofilm.Results:Bazheng Powder can not inhibit the growth of UPEC at a concentration of 400mg/m L,which proves that Bazheng Powder has no direct antibacterial ability against UPEC.Bazheng Powder at a concentration of 8 mg/m L cannot affect the growth rate of UPEC.In the bacterial gliding motility experiment,Bazheng Powder at a concentration of1 mg/m L can inhibit the spread of UPEC on the soft agar plate.The CCK-8 method showed that Bazheng Powder had no cytotoxicity to T24 cells when the concentration was below 1mg/m L.In adhesion and invasion experiments,Bazheng Powder at concentrations of 100 μg/m L,500 μg/m L and 1 mg/m L can prevent and inhibit the adhesion and invasion of UPEC to T24 cells in a concentration-dependent manner.The higher the concentration,the stronger the inhibition ability.Transmission electron microscopy showed that after adding Bazheng Powder at a concentration of 1 mg/m L,the fimbriae and flagella structure of UPEC were destroyed,and the surface of the bacteria became smooth.The results of crystal violet staining and confocal laser scanning microscope that Bazheng Powder at the concentration of 500 μg/m L and 1 mg/m L can inhibit the formation of UPEC biofilm in the urinary tract,and it is in a concentration-dependent manner.The higher the concentration,the stronger the inhibition ability.Conclusion: Bazheng Powder has no direct antibacterial effect on UPEC,and can inhibit the gliding motility of UPEC on a soft agar plate.At lower than the cytotoxic concentration,Bazheng Powder can prevent and inhibit the adhesion and invasion of UPEC to bladder epithelial cells,destroy the fimbriae and flagella structure of UPEC,and inhibit the UPEC biofilm formation.2 The mechanism of Bazheng powder in inhibiting the adhesion and biofilm of uropathogenic Escherichia coliObjective: Based on the transcription level from the two aspects of UPEC and host cells to explore the potential mechanism of Bazheng Powder in inhibiting the adhesion and biofilm of UPEC.Methods: Transcriptomics method was used to detect the influence of Bazheng Powder on the whole genes of UPEC.Then use RT-q PCR to detect the effect of Bazheng Powder on UPEC fimbriae and biofilm-related genes,and use RT-q PCR to detect the effect of Bazheng Powder on the expression of urothelial cell adhesion-related receptor genes.Results: In the transcriptomics results,compared with the control group,the Bazheng Powder group had 601 differentially expressed genes,of which 316 were up-regulated genes and 285 were down-regulated genes.Analysis of the enrichment results of differentially expressed genes showed that the main action pathways of Bazheng Powder on UPEC include:carbohydrate catabolic process,phosphotransferase system,biofilm formation,fructose and mannose metabolism.The RT-q PCR analysis was compared with the transcriptomics results,and the gene expressions of Fim A,Fim C,Fim D,Fim E,Fim F,Csg A,Csg F,Pap F were basically consistent with the transcriptomics results.In addition,Bazheng Powder can reduce the expression of bladder epithelial cell adhesion receptors Integrin α3,Integrin β1 and uroplakins UPIa and UPIb.Conclusion: Bazheng Powder can inhibit the expression of UPEC fimbriae genes.The transcriptomics results of UPEC suggest that Bazheng Powder can activate the phosphotransferase system,biofilm formation,fructose and mannose metabolism and other pathways to inhibit the adhesion of UPEC and the formation of biofilms.Bazheng Powder can inhibit the expression of urothelial adhesion receptors,improve host cell defense capabilities,and inhibit the adhesion of UPEC.3 Therapeutic effect of Bazheng powder on animal models of urinary tract infectionObjective: Construct the mouse model of urinary tract infection,study the therapeutic effect of Bazheng Powder on urinary tract infection in mice,and evaluate the therapeutic effect of Bazheng Powder on urinary tract infection in vivo experiments.Methods: A mouse model of urinary tract infection was constructed.After grouping,different concentrations of Bazheng powder were administered by gavage.After 5 days of treatment,the mortality and weight changes of the mice in each group were counted,and the changes in the number of bacteria in the urine on the first 1,3,and 5 days of administration were evaluated by the flat colony counting method.After the mice in each group were sacrificed,the flat colony counting method was used to evaluate the differences in the number of bacteria in the bladder and kidney of the mice in each group after 5 days of treatment.The morphological differences of the bladder and kidney of each group of mice were observed by naked eyes and photographed.Then,the effect of Bazheng Powder on the pathology of the bladder and kidney of mice with urinary tract infection was evaluated by HE staining.Results: Compared with the model group,after 5 days of treatment with Bazheng Powder,the mortality of mice was reduced.The average body weight of mice in the low-dose group of Bazheng powder was higher than that of the model group,and the weight of mice in the middle-dose group and high-dose group of Bazheng powder was lower than that of the model group.The bacterial counts in urine,bladder and kidney of mice in Bazheng Powder high,middle and low dose groups were lower than those in the model group.The bladder congestion of the mice in the model group was observed with naked eyes.The bladder congestion of the mice in the Bazheng powder group was significantly less than that in the model group,and the kidney morphology of the mice in each group did not change significantly.Compared with the model group,the pathological damage of the bladder and kidney of the mice in the Bazheng powder group was significantly reduced.Conclusion: Bazheng Powder can reduce the mortality of mice with urinary tract infection,reduce the number of bacteria in urine,bladder and kidney of mice with urinary tract infection,and improve the pathological damage of bladder and kidney in mice with urinary tract infection.4 The mechanism of Bazheng powder on animal models of urinary tract infectionObjective: From the perspective of the host of the urinary tract,the treatment targets and biological pathways related to urinary tract infection were screened out,and then the mechanism of Bazheng Powder’s treatment of urinary tract infection was verified in vivo and in vitro.Methods: Using bioinformatics methods to comprehensively analyze the omics results in the online database to find out the targets and biological pathways related to urinary tract infections.In animal experiments,ELISA was used to detect the expression of TNF-α,IL-1β,and IL-6 in mouse bladder and kidney tissues,and western blotting was used to detect NF-κB and integrin α3 and β1 in mouse bladder tissue.In cell experiments,construct NF-κB p65 overexpression vector,and then detect the effect of Bazheng Powder on TNF-α,IL-1βand IL-6 after overexpression of p65.Use western blotting to detect the expression of host adhesion receptor integrin α3,β1 and uroplakins UPIa,UPIb after overexpression of p65.After overexpression of p65,the effects of Bazheng Powder on the expression of host adhesion receptor integrins α3,β1 and uroplakins UPIa and UPIb were tested.Results: Bioinformatics analysis showed that TNF-α,IL-1β,IL-6,and NF-κB are the key targets of urinary tract infection,and the TNF signaling pathway is the main pathway of enrichment.In animal experiments,compared with the model group,the levels of TNF-α,IL-1β,and IL-6 in the bladder and kidney of mice in each dose group of Bazheng powder were significantly reduced.Compared with the model group,the protein expression of NF-κB in the Bazheng powder high-dose group was significantly reduced,and the expression of integrin α3 and β1 was significantly reduced.In cell experiments,the levels of TNF-α,IL-1β,and IL-6 were significantly increased after overexpression of NF-κB p65,and the levels of TNF-α,IL-1β,and IL-6 were significantly reduced after the treatment of Bazheng powder.Overexpression of NF-κB p65 promotes the expression of integrin α3 andβ1,and Bazheng powder can reduce the expression of integrin α3 and β1.Overexpression of NF-κB p65 promotes the expression of uroplakins UPIa and UPIb,and Bazheng powder can reduce the expression of uroplakins UPIa and UPIb after the effect.Conclusion: In urinary tract infections,inflammation is closely related to the adhesion of UPEC.Bazheng Powder can reduce the expression of adhesion receptors related to UPEC by reducing the inflammation level of urinary tract infections.So as to inhibit the adhesion of UPEC to the host,and achieve the effect of treating urinary tract infections..